This invention is in the field of plant biotechnology. More specifically, the invention relates to a mutant barley lipoxygenase 1 gene (lox-1) that encodes an enzyme with severely reduced 9-hydroperoxy-octadecadienoic acid forming activity. The invention also relates to the use of barley cultivars homozygous for mutated lox-1 in brewing processes to reduce the formation of off-flavors in brewed products, such as beer, during storage.
Lipoxygenases are a family of enzymes (EC 1.13.11.12) that catalyze the dioxidation of free and esterified poly-unsaturated fatty acids containing a 1(Z), 4(Z)-pentadiene configuration. The products of lipoxygenase-catalyzed reactions have long been suspected as major culprits for the appearance of stale flavors in plant grain/seed and grain/seed derived food products (Robinson et al., 1995, Food Chem., 54: 33-43). Lipoxygenases have been implicated in the production of volatile hexanal aldehydes generated during soybean processing, which have an undesirable aroma, limiting the use of soybean proteins in food products. Three lipoxygenase isozymes expressed in soybean seed are believed to contribute to lipid oxidation and hexanal formation. Soybean mutants lacking one or more of these isozymes have been generated with the aim of reducing hexanal formation and improving their flavor stability. The success of this approach has been evaluated by Hildebrand et al., 1990, J. Agric. Food Chem. 38: 1934-1936. Mutants lacking soybean lipoxygenase 3 produced higher hexanal levels, suggesting that this isozyme diverts 13-hydroxyperoxyoctadecanoids, produced by lipid oxidation, towards non-volatile products. The field performance of triple-null soybean lines, lacking all three seed lipoxygenases, has shown that these enzymes are not essential for normal agronomic and seed characteristics (Narvel et al., 1998, Crop Sci. 38: 926-928).
Lipoxygenases have also been implicated in the generation of off-flavors in rice, which can occur during grain storage. The release of free fatty acids can be detected in stored grain, which is indicative of the metabolism of the triglyceride reserves. The rice variety Daw Dam was found to accumulate lower levels of pentanals and hexanals giving a better flavor stability on storage (Susuki et al., 1999, J. Agric. Food Chem., 47: 1119-1124). This desirable phenotype was attributed to the absence of rice lipoxygenase-3, which oxidizes unsaturated lipid acyl chains to form 9-hydroperoxyoctadecadienoic positional isomers.
It is recognised that the lipoxygenase pathway is complex with many branches and its role in numerous aspects of plant growth and physiology are not fully understood. Modifications of the lipoxygenase pathway which alter 9-hydroperoxidation activity in seed crops are proposed to regulate their susceptibility to mycotoxin contamination by Aspergillus spp. (WO 9726364), which is consistent with the involvement of this pathway in plant pathogen resistance, but is not related to the aims of the invention herein described.
Among the many aroma volatiles which contribute to the flavor of beer, the higher unsaturated aldehydes with a 6-12 carbon chain have particularly low organoleptic flavor thresholds (Meilgaard 1975, MBAA Tech. Quart. 12: 151-168). Trans-2-nonenal, which is a member of this group, has both an extremely low flavor threshold of 0.11 ppb and contributes an unpleasant straw-like, xe2x80x9ccardboardxe2x80x9d flavor to the beer. The characteristic off-flavor caused by trans-2-nonenal is a common characteristic of beers stored for 1-3 months or more and is particularly detrimental to the flavor of lager beer, which is brewed with light malts and has a delicate flavor.
Sulfite has long been known to improve the flavor stability of beer, not only by binding oxygen and acting as an anti-oxidant, but also by forming volatile bisulfite addition compounds with aldehydes and ketones present in the beer. The two major sources of sulfite in beer are sulfite produced by yeast during fermentation via the sulfur assimilation pathway and sulfite added to the beer prior to packaging. Fermentation conditions that enhance yeast sulfite production and secretion will allow the formation of sulfite-carbonyl adducts from carbonyls present in the wort and prevent their further metabolism by the yeast (Dufour 1991, Proc.Eur. Brew. Conv. Congr., Lisbon, pp. 209-216). In this manner carbonyls such as acetaldehyde and diacetyl may be transferred to the beer. The ability of sulfite to prevent the appearance of the carbonyl compound trans-2-nonenal during beer aging has been demonstrated by brewing beer with a yeast strain blocked in the sulfur assimilation pathway (Johannesen et al., 1999, Proc.Eur. Brew. Conv. Congr., Nice, pp. 655-662). Following bottling, the beer was subjected to forced aging by storing it at 37xc2x0 C. for 7 days, after which trans-2-nonenal levels were found to be well above the taste-threshold. If 10 ppm sulfite was added to the low-sulfite beer just prior to bottling, the appearance of trans-2-nonenal during forced aging was significantly reduced. The reaction between sulfite and carbonyl compounds is reversible and under thermodynamic and kinetic control. The apparent equilibrium constants for bisulfite compounds ranges from 10xe2x88x926 M for carbonyl compounds such as acetaldehyde, hexanal, and decanal, to 10xe2x88x923 for diacetyl and pyruvate (Dufour 1991, supra). During beer storage, gas exchange through the packaging will allow oxygen into the beer and sulfite will be lost, such that weaker bisulfite adducts will dissociate, allowing free carbonyls to appear in the beer. While sulfite unquestionably enhances the flavor-stability of beer, particularly in the short-term, its retention in packaged beer is strongly dependent on gas exchange through the packaging and temperature. In a finished beer the natural levels of sulfite produced during fermentation are variable and the addition of sulfite prior to bottling is not a universally accepted practice. For these reasons sulfite alone does not provide a reliable method to enhance the long-term flavor-stability of beer under the different beer storage conditions used around the globe.
It is generally accepted that the trans-2-nonenal found in beer results from the oxidation of polyunsaturated fatty acids derived from barley grain lipids, where the 18-carbon chain fatty acid, linoleic acid [classified as an 18:2,n-6 polyunsaturated fatty acid (Broun, Gettner and Sommerville 1999, Annu. Rev. Nutr. 19: 197-216)] is the most abundant. However, there is little agreement in the literature as to the mechanism whereby trans-2-nonenal is formed. The presence of enzymatic pathways leading to trans-2-nonenal formation from poly-unsaturated fatty acids has been proposed, but the individual enzymatic steps have never been demonstrated experimentally in barley grain or during the malting process (Gardner 1988, Adv. Cereal Sci. Technol. 9: 161-215). The concept of using anti-sense or co-suppression gene technology to reduce lipoxygenase-1 levels in barley grain, and thereby control 9-hydroperoxidation and reduce aldehyde and alcohol levels in the finished barley grain, has been proposed as a means to control off-flavor formation, but results of such an approach are not reported (McElroy and Jacobsen, 1995, Bio/Technology 13: 245-249).
A forcing test has been developed as a method for assessing the trans-2-nonenal potential of a beer, where trans-2-nonenal formation in wort or beer is induced by subjecting samples to elevated temperatures at reduced pH, (100xc2x0 C., at pH 4.0 for 2 hours). Attempts to correlate the trans-2-nonenal potential in wort and finished beer with the total level of lipoxygenase activity in the kilned malt have indicated that lipoxygenase may contribute to the appearance of trans-2-nonenal in aged beer (Drost et al., 1990, J. Am. Soc. Brew. Chem. 48: 124-131). The conclusions that can be drawn from this study, however, are severely limited by the fact that the lipoxygenase activity in the barley malt was regulated at the end of the malting process by the degree of enzyme inactivation during kiln drying. Thus, only the effect of the residual malt lipoxygenase activity on the trans-2-nonenal potential in the derived wort and finished beer was examined. The study failed to evaluate the lipoxygenases that catalyse the first step in the lipoxygenase enzymatic pathway in the barley grain during development and malting, and their role as determinants of trans-2-nonenal levels found in beer. Indeed, the absence of barley cultivars deficient in one or more lipoxygenase isoenzyme has made it impossible to provide convincing evidence for the role of the lipoxygenase pathway in barley malt in controlling the formation of trans-2-nonenal. Such experiments are needed to evaluate the contribution of enzymatic, as compared to auto-oxidative/chemical pathways, to the formation of trans-2-nonenal in beer. The brewing process involves a high temperature step of wort boiling where these non-enzymatic reactions are proposed to occur (Noxc3xal et al., 1999, J. Agric. Food Chem. 47: 4323-4326).
This invention provides a barley cultivar having greatly reduced lipoxygenase-1activity. In one embodiment, the barley plants of the invention contain a mutant lox-1 gene expressing greatly reduced levels of the isoenzyme lipoxygenase-1. In an alternative embodiment, the barley plants contain a heterologous nucleic acid sequence expressing an antisense sequence to the wild-type lox-1, thereby reducing the enzyme""s activity.
As shown herein, malt and wort produced from the reduced lipoxygenase barley of the invention, for example, from barley cultivars homozygous for a mutant lox-1 gene, are useful to produce beer with significantly enhanced flavor stability and reduced trans-2-nonenal levels, particularly under conditions known to promote the appearance of T2N. The invention demonstrates a correlation between the activity of barley malt lipoxygenase-1 to produce 9-hydroxyperoxy-octadecadienoic acids (9-HPOD), and the presence of trans-2-nonenal in beer. The invention further demonstrates that the use of barley homozygous for the mutant lox-1 gene in the brewing process improves the flavor stability of the beer, both during storage and on exposure to elevated storage temperatures. These properties enhance the quality of the beer, and are useful to extend its shelf-life and reduce the need to cool beer during transport and storage.
The invention provides barley plants and portions thereof having reduced lipoxygenase-1 activity, including barley plants expressing mutant LOX-1 protein as described herein, as well as methods for producing such barley plants, plant portions, products of the plants, and particularly malt and beer products produced from the barley plants of the invention.